SICA-mediated cytoadhesion of Plasmodium knowlesi-infected red blood cells to human umbilical vein endothelial cells

Zoonotic malaria due to Plasmodium knowlesi infection in Southeast Asia is sometimes life-threatening. Post-mortem examination of human knowlesi malaria cases showed sequestration of P. knowlesi-infected red blood cells (iRBCs) in blood vessels, which has been proposed to be linked to disease severity. This sequestration is likely mediated by the cytoadhesion of parasite-iRBCs to vascular endothelial cells; however, the responsible parasite ligands remain undetermined. This study selected P. knowlesi lines with increased iRBC cytoadhesion activity by repeated panning against human umbilical vein endothelial cells (HUVECs). Transcriptome analysis revealed that the transcript level of one gene, encoding a Schizont Infected Cell Agglutination (SICA) protein, herein termed SICA-HUVEC, was more than 100-fold increased after the panning. Transcripts of other P. knowlesi proteins were also significantly increased, such as PIR proteins exported to the iRBC cytosol, suggesting their potential role in increasing cytoadhesion activity. Transgenic P. knowlesi parasites expressing Myc-fused SICA-HUVEC increased cytoadhesion activity following infection of monkey as well as human RBCs, confirming that SICA-HUVEC conveys activity to bind to HUVECs.

www.nature.com/scientificreports/ prevents the clearance of iRBCs by the spleen and serves as a major virulence factor 8 . Sequestration of P. knowlesi-iRBCs in blood vessels was observed in a post-mortem examination of humans and monkeys 9,10 . Abdominal pain was found to be a risk factor for severe knowlesi malaria 11 , for which gut ischemia due to accumulation of iRBCs in the blood vessels was suggested as a cause 6 . Schizont Infected Cell Agglutination (SICA) proteins have been proposed to have a role in the cytoadhesion of P. knowlesi-iRBCs 12 ; however, this interaction is not well characterized. SICA protein is a type I transmembrane protein encoded by a SICAvar multigene family with at least 136 members 13 . This protein has multiple cysteine-rich domains (CRDs) in the extracellular region, and an intracellular region which shares homology with Plasmodium molecules expressed on the RBC membrane, such as P. falciparum PfEMP1 and SURFIN family members 12,14 . In addition to SICA protein, P. knowlesi expresses KIR proteins, encoded by the pir gene family which is widely represented in the rodent and primate malaria parasites, and for which binding activity of the P. vivax homolog VIR was reported 15 .
To gain insights into the mechanism of sequestration in P. knowlesi-iRBCs in humans, we aimed to identify molecules responsible for the cytoadhesion of P. knowlesi-iRBCs to human endothelial cells. We repeatedly panned P. knowlesi-iRBCs against human venous endothelial cells to enrich cytoadhesive iRBCs. RNA-seq analysis of parasites with cytoadherence activity identified one SICA open reading frame (ORF) as a strong candidate, and following a transfection experiment confirmed that the identified SICA was capable of increasing the cytoadhesive activity of transfected parasites.

Cytoadhesion of P. knowlesi-iRBCs against human umbilical vein endothelial cells (HUVECs)
is unstable. To evaluate if P. knowlesi-iRBCs could bind to human endothelial cells, we repeatedly panned monkey RBCs infected with a wild-type P. knowlesi on HUVECs. The first selection experiment (exp-1) was initiated with two wells, but one well was contaminated; thus, only one sample before the panning selection ("pre-1" sample) was used to compare with samples after the 6th and 8th pans. Bound iRBCs were not detected at the beginning, but we observed iRBCs bound on the HUVECs from the 3rd pan onward, and with each panning the number of bound RBCs gradually increased (Fig. 1A). RNA samples were obtained before ("pre-1") and after the 6th and 8th pans from one well ("6th pan" and "8th pan", respectively). When the culture was maintained without panning selection for 88 days after the 10th pan, the binding activity became undetectable. The increased binding activity was repeatedly observed when the culture was panned, which was reduced if the panning selection was not performed (Fig. S9A). For the second selection experiment (exp-2), 2 wells were simultaneously panned against HUVECs, and bound iRBCs were seen after the 5th pan and the number of bound RBCs increased with each panning (Fig. 1B). RNA samples were prepared before ("pre-3") and after the 13th pan ("well-1" and "well-2"). After the 13th pan, we further maintained these 2 parasite lineages without panning selection and examined their cytoadhesion activity on days 17, 34, 42, and 53 after the 13th pan. We found that the cytoadhesion activity of the two lineages gradually decreased and the level became similar to the level before panning selection on day 53 after the 13th pan was performed (Fig. 1C). These results suggest that the cytoadhesion property of P. knowlesi-iRBCs was unstable.
Transcripts of two SICAvar fragments significantly and reproducibly increased after panning selections. RNA samples were collected at 4 h (ring form early trophozoite), 8 h (late trophozoite), and 24 h (schizont) after RBC invasion and were subjected to RNA-seq analysis. The transcript expression fold change was obtained by dividing the values after panning by the values before panning. Firstly, we assessed the correlation among samples. The scatter plot of the log2 fold change between the 6th and 8th pans of exp-1 against pre-1 revealed that these two samples were positively correlated with a moderate ρ value (0.427) ( Fig. 2A, left). A positive correlation was also observed between wells 1 and 2 of the exp-2 13th pan against pre-3 (ρ = 0.784, Fig. 2A, middle). However, there was no positive correlation between exp-1 and exp-2 ( Fig. 2A, right for exp-1 8th pan against pre-1 and exp-2 13th pan well-1 against pre-3; Figs. S1 and S2 for other combinations), suggesting that if the culture was handled simultaneously, the transcription pattern was similarly changed, but if the culture process was independent, the change of the transcription pattern differed. This observed difference between exp-1 and exp-2 is not due to the inclusion of the ORFs with low coverage, because the analysis excluding ORFs for which the average FPKM of all data was less than 10 also showed the same tendency (Figs. S3 and S4). The independent panning selections both identified two ORFs, PKNH_0814200 (type II SICA) and PKNH_0814300 (type I SICA), that increased their transcript expression ( Fig. 2A, red and blue dots, respectively).
Data from the 8th pan of exp-1 and the 2 wells (well-1 and well-2) of exp-2 13th pan were combined, and the log2 fold change of each ORF and the significance (− log 10 q) were visualized by volcano plots (Fig. 2B). The data of the exp-1 6th pan was excluded because the samples after the 6th and 8th pans were collected from the same culture lineage. Volcano plots indicated that PKNH_0814200 and PKNH_0814300 described above were the most differentially and highly expressed ORFs at all 3-time points. Tables 1 and 2 summarize ORFs for which the average FPKM of all data was at least 10, the mRNA expression was at least twofold increased (Table 1) or decreased (Table 2) after the panning by the analysis of the combined data, q value < 0.05, and consistently increased or decreased in all 4 test samples (exp-1 6th and 8th pans and exp-2 13th pan wells 1 and 2). In addition to PKNH_0814200 and PKNH_0814300, the transcripts of 2 PIR proteins (PKNH_0312000 and PKNH_0807800), which are predicted to be exported to the RBC cytosol, were increased consistently throughout the samples at the schizont stage (24 h). Consistently decreased ORFs included 3 SICA-related proteins (PKNH_1144600, PKNH_0727400, and PKNH_1144500) and proteins exported to the RBC cytosol (PKNH_1401700 and PKNH_1357100). When a less stringent criteria was used with a P value less than 0.05, 16 differentially expressed ORFs with absolute log2 fold change > 2 and P value < 0.05 at 4 h, 8  We then evaluated whether ORFs whose transcript levels increased with the increase in cytoadhesion activity would decrease with the loss of adhesion activity. To this end, we conducted RNA-seq analysis against P. knowlesi samples (both well-1 and well-2) at 53 days after the 13th pan in experiment 2 described above. Samples from both wells showed that FPKM values of PKNH_0814200 and PKNH_0814300 decreased at all three time points (Table S4). In addition, we found increased FPKM values of PKNH_0312000 and PKNH_0807800 (both are PIR proteins) with increasing cytoadhesion activity decreased with loss of the adhesion activity; whereas decreased FPKM values of PKNH_1144500 and PKNH_1144600 (both are SICA proteins) with increasing cytoadhesion activity increased with loss of the adhesion activity.
Two SICAvar fragments annotated in PlasmoDB belong to one ORF frame. We focused on ORFs PKNH_0814200 and PKNH_0814300 because they were changed most highly and significantly compared to the other ORFs. Examination of the genome sequence information of P. knowlesi H strain in the PlasmoDB database revealed that PKNH_0814200 and PKNH_0814300 are adjacent within the genome, but sequence between . RNA samples were obtained before and after the 6th and 8th pans (blue circle on the x-axis). Images after the 1st, 4th, and 7th rounds are shown below. (B) Second panning selection experiment (exp-2). Two wells (well-1 and well-2) were simultaneously panned 13 times and RNA samples are collected before and after the 13th pan (blue circle on the x-axis). Images after the 1st, 7th, and 13th rounds are shown below. (C) Binding activity of iRBCs in 2 wells at 17, 34, 42, and 53 days after the 13th pan of exp-2 without panning selection pressure. www.nature.com/scientificreports/ the two ORFs is lacking. Because PKNH_0814300 encodes the N-terminal side and PKNH_0814200 encodes the C-terminal side of a predicted SICA protein, we evaluated the genome information for another line of P. knowlesi (Malayan strain PK1 A) in PlasmoDB and found that the two H strain ORFs are annotated as a single ORF (PKNOH_S100042200). To this end, we determined the cDNA sequence in the H strain and found that PKNH_0814200 and PKNH_0814300 indeed represent one ORF consisting of 12 exons (Fig. 2C). We named this gene sica-huvec and the encoded protein SICA-HUVEC. Experimentally obtained cDNA sequence was www.nature.com/scientificreports/ deposited into the DDBJ database (LC663824). SICA-HUVEC is a type I transmembrane protein composed of one conserved head domain and seven SICA cysteine-rich domains in its extracellular region, followed by one transmembrane domain and a cytoplasmic region (Fig. 2D).

Establishment of a transgenic P. knowlesi parasite line expressing SICA-HUVEC-Myc.
To validate if SICA-HUVEC was responsible for the cytoadhesion to HUVECs, a transgenic P. knowlesi line was established with an episomal plasmid expressing the full-length SICA-HUVEC fused with 2 Myc epitopes at its C-terminus (SICA-HUVEC-Myc) (Fig. 3A). To assess the expression of SICA-HUVEC-Myc, proteins were sequentially extracted from transfectant-infected monkey RBCs by repeated freeze-thaw (Fz); with a nonionic detergent, Triton X-100 (Tx); and then an ionic detergent, SDS. Western immunoblotting with anti-Myc antibody revealed a major band of approximately 250 kDa in all Fz, Tx, and SDS fractions of the transfectant expressing SICA-HUVEC-Myc; which was similar to the calculated molecule weight of 230 kDa based on the amino acid sequence (Fig. 3B). SICA-HUVEC-Myc was not fully extracted with Tx, suggesting an association with detergent-resistant membrane; which is similar to the extraction pattern of P. falciparum PfEMP1. No band was detected from the SDS extract of the wild-type P. knowlesi, indicating that the bands observed for the transfectant were from exogenously expressed SICA-HUVEC-Myc protein.
When iRBCs were treated with trypsin, the intensity of the ~ 250-kDa band was significantly reduced and bands at approximately 50 kDa and 24 kDa were detected in the trypsin-treated sample, but not in the untreated Table 1. Identified P. knowlesi ORFs whose transcript expression was significantly increased. ORFs satisfying the following criteria are shown: (1) whose average FPKM of all data was at least 10, (2) average fold change values were at least 4, and (3) q value is less than 0.05. P and q values were obtained excluding the exp-1 6th pan data.

ID Annotation
Log2 fold change after panning  Table 2. Identified P. knowlesi ORFs whose transcript expression was significantly decreased. ORFs satisfying the following criteria are shown: (1) whose average FPKM of all data was at least 10, (2) average fold change values were less than or equal to 4, and (3) q value is less than 0.05. P and q values were obtained excluding the exp-1 6th pan data. PKNH_1144500 was annotated as "Protein conserved in P. knowlesi" in PlasmoDB, but identified as a SICA fragment by BLASTP analysis.

ID Annotation
Log2 fold change after panning  www.nature.com/scientificreports/ sample (Fig. 3C). The size of the 24-kDa band was consistent with the expected size of the cleaved product after K 1876 (25 kDa). The intensity of the control EXP2 protein was similar between trypsin-treated and untreated samples. These results suggest that SICA-HUVEC-Myc is exposed on the surface of iRBCs. IFA with anti-Myc antibody yielded a dotted pattern within the iRBC cytosol for all parasite stages examined (Fig. 3D), consistent with the export of SICA-HUVEC-Myc and localization at Sinton Mulligan's clefts-membranous structures in the P. knowlesi-iRBC cytosol where SBP1 and MAHRP2 are localized 16,17 . Giemsa-staining after IFA confirmed SICA-HUVEC-Myc co-localized with Sinton Mulligan's stipplings (Fig. 3E). Although signals were not clearly seen on the iRBC membrane, the IFA results suggested trafficking of Plasmodium molecules destined for exposure on the iRBC membrane.
Transgenic parasites expressing SICA-HUVEC-Myc in monkey, as well as human RBCs, showed higher cytoadhesion activity to HUVECs. The HUVEC cytoadhesion activity was evaluated for RBCs infected with the obtained transfectant parasites. In addition to the wild-type parasite, a transfectant control was generated expressing mCherry in place of SICA-HUVEC-Myc ( Fig. 4 and Fig. S7). Independent experiments using monkey RBCs consistently showed significantly higher binding of the SICA-HUVEC-Myc expressing transgenic parasite line than the two controls. The binding activity between the wild-type and mCherry-expressing transgenic parasites was not significantly different, excluding the possibility that transfection procedures affected the result (Fig. 4A,B). Together, these results indicated that P. knowlesi gained binding activity to HUVECs following the expression of SICA-HUVEC-Myc. We then evaluated if the cytoadhesion activity can be seen when human RBCs (hRBCs) were infected with the transfectant parasite line. Although long-term cultivation with hRBCs is not possible, the parental P. knowlesi H-DMU line can invade into and multiply within hRBCs for two cycles. We took advantage of this and let the purified schizonts of two transfectant lines invade into hRBCs, then the cytoadhesion activity of infected hRBCs was examined. We confirmed that approximately 95% of the prepared parasite-infected RBCs were hRBCs by differentiating hRBCs from monkey RBCs using an anti-human glycophorin A antibody (Fig. S8). Independent binding experiments using hRBCs consistently showed that the number of bound hRBCs infected with SICA-HUVEC-Myc-expressing parasites was significantly higher than that of hRBCs infected with mCherry-expressing parasites (Fig. 4B).

Discussion
In this study, to the best of our knowledge we show for the first time that P. knowlesi parasites are able to change the cytoadhesion activity of their infected RBCs. We identified a SICA protein, termed SICA-HUVEC, which can mediate the cytoadhesion of HUVECs by infected monkey as well as human RBCs. Correspondingly, when iRBCs lost their cytoadhesion activity, the transcript level of SICA-HUVEC also decreased. In addition to SICA-HUVEC, RNA-seq analysis showed that transcripts for two PIR proteins were significantly increased in the cytoadherent parasites, suggesting that these proteins might be additively or synergistically involved in the cytoadhesion. www.nature.com/scientificreports/ RBCs infected with transgenic P. knowlesi expressing Myc-tagged SICA-HUVEC did not bind as high as the naturally selected HUVEC-binding lines. qRT-PCR revealed that a similar amount of the transcripts for the putative ligand were detected from the transgenic line compared to the naturally selected parasites (Fig. S9B,C); indicating that the transcript levels were not the determinant of the difference in the cytoadhesion activity. Two possibilities can be considered; firstly, the introduced Myc-tagged SICA-HUVEC may not be exposed on the surface of iRBCs as efficiently as the natural SICA-HUVEC, perhaps due to the addition of the Myc-epitope to its C-terminus. The amino acid linker sequence between SICA-HUVEC and the Myc-epitopes is GTELL-DGP. Glycine and proline are helix breaker residues that enable movement of the following domain freely from potential physical restriction by the upstream domain structure. We have used the same linker sequence to fuse Myc-epitopes to other Plasmodium proteins, such as P. knowlesi SBP1 and P. knowlesi KAHRP2, for which Myctags were detectable by immunoelectron microscopy; indicating that this linker sequence was at least able to expose fused Myc-epitope to be recognized by the anti-Myc antibody 16,17 . However, the molecular mechanism to translocate plasmodial molecules to the surface of the iRBC is not well understood, thus any optimized linker sequence has not yet been reported for this process.
Secondly, other alterations might be required for the higher binding; for example, we found that the transcript levels of several P. knowlesi ORFs encoding proteins exported to the iRBC cytosol were consistently changed in addition to sica-huvec (Tables 1, 2; Tables S2 and S3), which might contribute to the increased cytoadherence activity of the naturally selected lines. We consider the latter scenario is likely, but further investigation is required to clarify this point.
To understand the biological significance of the cytoadhesion, it is important to identify both the parasite ligand and the host receptor. Multiple host vascular receptors have been identified for P. falciparum PfEMP1, such as CD36, ICAM1, VCAM1, and chondroitin sulfate A 18 21 to human endothelial cells (human cerebral microvascular endothelial cells, human pulmonary microvascular endothelial cells, or human renal glomerular endothelial cells) stimulated with P. knowlesi culture supernatant, and which was inhibited by antibodies against chondroitin sulfate proteoglycan 4 (CSPG4), ICAM1, VCAM1, PECAM1, P-selectin, or CD36 22 . Thus, these molecules are receptor candidates of these cells for cytoadhesion of P. knowlesi-iRBCs. As for HUVECs, comprehensive proteomics analysis revealed that of the above mentioned 6 molecules the highest expressions observed were for PECAM1 (Peptide spectrum match (PSM) = 431), followed by ICAM1 (PSM = 59), and low expression of VCAM1 (PSM = 0.21); whereas CSPG4, P-selectin, and CD36 were not detected 23  To enrich schizont stage parasites, P. knowlesi cultures were used having more than 1% parasitemia of schizonts. The iRBCs were suspended in 5 mL of incomplete culture medium (ICM), which does not contain AlbuMax II, sodium bicarbonate, and gentamicin; then layered above a 50% Nycodenz solution (1.077 g/mL) and centrifuged at 900×g for 12 min at 20 °C 17 . The schizont-enriched layer was then collected, and the cells were washed twice with ICM. The schizont-iRBCs were confirmed by Giemsa-staining of blood smears.
For binding assays, HUVECs were seeded and grown to high confluency on the bottom of poly-l-lysinecoated 6-well plates where poly-l-lysine-coated coverslips (13 mm ø, thickness 0.13-0.17 mm; Matsunami Glass, Osaka, Japan) were placed. Before use HUVECs were rinsed 3 times with 3 mL of pre-warmed Basal Medium (BM; C-22210; PromoCell). P. knowlesi parasites were cultured at a 5 mL scale until the parasitemia of trophozoite and schizont stages became more than 3%, washed with 5 mL of pre-warmed BM, adjusted for parasitemia to approximately 3.5%, resuspended in 900 μL of BM (final concentration of iRBCs was 17-19%), and then placed on HUVECs in triplicate wells for each sample (300 μL/well). The cells were incubated at 37 °C for 75 min with a www.nature.com/scientificreports/ 5% O 2 , 5% CO 2 , and 90% N 2 gas mixture, with gentle shaking to suspend the iRBCs at 30 and 60 min during the incubation. After incubation, coverslips with HUVECs covered with RBCs were gently removed, washed with BM, fixed with 1% glutaraldehyde at RT for 30 min, and then stained with Giemsa's solution. The images were observed by light microscopy and digitally captured, then the numbers of iRBCs per 100 HUVECs were counted. The difference between the test and control groups was examined by one-way ANOVA followed by post-hoc Tukey's multiple comparison test or two-tailed Student's t-test. We used HUVECs passaged less than 10 times because it was reported that HUVECs passaged more than 15 times begin to lose their cell spreading activity 24 . For the panning selection, the iRBCs remaining on HUVECs after removing the coverslips were washed 5 times with 3 mL of BM, and then 3 mL of P. knowlesi CM containing 2% uninfected rhesus monkey RBCs was added. The parasites in the 6-well plates were incubated with the above gas mixture overnight at 37 °C, then RBCs infected with newly invaded parasites were recovered and cultured in new flasks in 5 mL of CM containing approximately 2% rhesus monkey RBCs.
RNA sequencing (RNA-seq). Total RNAs were prepared using the TRIzol reagent. For exp-1, libraries were generated using a TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA), and then 150 bp paired-end reads were sequenced on a HiSeq2500 (Illumina). For exp-2, libraries were constructed using a Next Ultra RNA Library Prep Kit for Illumina (New England Bio Labs, Ipswich, MA, USA) and 150 bp paired-end reads sequenced on a Novaseq 6000 (Illumina). The reads obtained by RNA-seq were mapped by TopHat (version 2.1.1) to the P. knowlesi H strain genome sequence acquired from PlasmoDB (release-31). Aligned reads per gene were counted by HTseq (version 0.6.1), and the ORFs for which the average FPKM of all data was less than 1 were excluded from further analysis. Differentially expressed genes were identified by DESeq2 (version 1.30.1) with their q values, which are adjusted P values obtained by considering the false discovery rate. Volcano plots and correlation plots were generated using Microsoft Excel, and Spearman's rank correlation coefficient (ρ), and significances were calculated using GraphPad Prism (version 7). cDNA synthesis, RT-PCR, and sequencing. Extracted RNA was treated with DNase I (Promega, Madison, WI, USA) and further purified using an SV Total RNA Isolation System kit (Promega). cDNA was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. PCR was done using KOD-Plus-Neo (Toyobo, Osaka, Japan) with primer sets SICA-HUVEC.  (Table S1). Amplified PCR products were directly sequenced or purified using NucleoSpin ® Gel and PCR Clean-up (Takara Bio, Kusatsu, Japan), ligated with pGEM ® -T Easy plasmid (Promega), and 2 clones were sequenced using M13F primer, M13R primer, and primers designed for sequencing (Table S1). Obtained sequences were assembled and deposited to the DDBJ database under the accession number LC663824.
Transgenic parasites. Synchronized P. knowlesi schizonts were transfected using Amaxa Nucleofector 4D (Lonza, Basel, Switzerland) and a P3 Primary cell 4D Nucleofector X Kit L (Lonza). Approximately 20 μg of plasmid DNA in double-distilled H 2 O was added to 100 μL of P3 primary cell solution. Approximately 5 × 10 7 purified schizonts were resuspended in the DNA plus P3 primary cell solution, transferred into cuvettes (Lonza), and electroporated using program FP158. The cuvettes were immediately put on ice, then the electroporated cells were transferred to tubes containing 500 μL of pre-warmed CM with 20% rhesus monkey RBCs. The mixture was incubated on a thermo shaker (Thermomixer Comfort; Eppendorf, Hamburg, Germany) at 37 °C at 1000 rpm for 2 h, then transferred to a tissue culture flask containing 4.5 mL of CM pre-warmed to 37 °C to a final hematocrit of 2%. WR99210 was supplied in culture medium to a final concentration of 1.25 nM at 24 h post-transfection. The drug concentration was increased two-fold once drug-resistant parasites appeared, and the transgenic parasites were ultimately maintained with 10 nM WR99210.

SDS-PAGE and Western blot.
RBCs infected with unsynchronized parasites were incubated with 0.15% saponin in PBS containing a protease inhibitor (PI) cocktail (cOmplete™ ULTRA mini EASYpack; Roche, Basel, Switzerland) (PBS-PI) at 4 °C for 3 min, washed once with PBS-PI, then frozen at − 80 °C until use. The watersoluble fraction (FT fraction) was obtained by freeze-thawing (FT) the pellets in PBS-PI 3 times between 4 °C